Project Summary/Abstract Multiple myeloma (MM) is an incurable cancer of terminally differentiated plasma cells (PC), characterized by abundant immunoglobulin (Ig) synthesis. Most PC are short lived and die only a few days after differentiation through unclear molecular mechanisms. MM pathogenesis also remain elusive with no single genetic driver mutation, but pervasive DNA damage. We have previously shown increased cargo of polyubiquitinated (polyUb) proteins and decreased proteasome activity in differentiating B lymphocytes and we hypothesized proteotoxicity as driver of PC death. By using absolute mass spectrometry (AQUA) to quantify ubiquitin (Ub) in the same B cell differentiation model, our preliminary data show that 60% free Ub depletion occurs in PC compared to resting B cells, concomitantly with maximal Ig secretion and apoptosis. Second, we discovered that cells surviving long term post differentiation can be identified and display markers of ongoing DNA damage. As Ub is necessary for proper DNA damage response (DDR), polyUb protein degradation and DDR compete for the same Ub pool. Based on our work, we propose a unifying model for short-lived PC death and MM pathogenesis centered on critical Ub depletion. Our core hypothesis is that in PC, proteotoxic stress secondary to sustained Ig synthesis, leads to free Ub depletion thus causing impaired DDR. Most PC will be unable to recover from this crisis, however a small subset of genomically unstable PC may survive, presumably due to accumulation of survival promoting mutations, constituting a premalignant state for MM. We further hypothesize that Ub gene Ubc may be a haploinsufficient tumor suppressor genes in B cell malignancies. Herein, the applicant, Dr. Giada Bianchi, presents a comprehensive plan to test these hypotheses articulated in 2 specific aims: (1) to probe a causative link between Ig synthesis, Ub depletion and apoptosis in PC; and (2) to evaluate whether Ub depletion is sufficient to cause genomic instability in PC and drive MM pathogenesis. Data gathered during the course of our investigation will provide novel data regarding the biology of normal and malignant PC and potentially uncover a novel oncogenic mechanism - functional depletion of Ub - with applicability to other malignancies and opportunity for innovative, molecularly targeted therapies. To this end, Dr. Bianchi has carefully selected a mentoring committee and collaborators who are world-renowned experts in Ub, MM, DDR, proteomics and B cell biology. Such team will provide knowledge, models and technologies to render the testing of Dr. Bianchi?s hypotheses feasible within the proposed 5-year frame. The applicant?s mentoring committee has furthermore designed a detailed career plan based on regular meeting, attendance of workshops, classes and international meetings which will further accelerate the trajectory of Dr. Bianchi to become an independent investigator in cancer biology in the next 5 years.